Project Summary/Abstract: Optogenetic methods for modulating neural activity are revolutionizing neurobiological research. Brief exposure to light of cells expressing channelrhodopsin-2 (ChR2) can elicit excitatory cation fluxes (or inhibitory ion fluxes with the halorhodopsin NpHR). The consequences of optogenetic stimulation would optimally be recorded by non-invasive optical methods. However, most current optical methods for monitoring neural activity are based on fluorescence excitation that can cause unwanted stimulation of the optogenetic probe and other undesirable effects such as tissue autofluorescence. Luminescence is an alternate optical technology that avoids the problems associated with fluorescence. A novel luminescence-based methodology for monitoring neural activity as a new tool for functional neuroscience will be developed in this project. Luminescence avoids the complications associated with fluorescence because it is an enzymatic reaction that circumvents the need for excitation. The newly developed luminescence sensors for neuronal activity are genetically encodable and can be targeted to specific cell types and to specific cellular loci that are involved in neural activity. These sensors respond to the calcium ion fluxes generated by neuronal activity by changing the intensity and/or spectrum of their luminescence emission. In the latter case, a novel sensor based on Bioluminescence Resonance Energy Transfer (BRET) is modulated by neural activity so that the spectrum of luminescent emission is altered when neurons are activated. This new luminescence methodology avoids the drawbacks of electrophysiology and fluorescence excitation (esp. off-target optogenetic stimulation, photobleaching & tissue autofluorescence), and therefore optimally partners with optogenetic methods for in vitro and in vivo stimulation. These luminescence sensors will be characterized in conjunction with optogenetic stimulation of neuronal activity in vitro, especially while actuating both ChR2 (excited by blue light) and NpHR (excited by orange light) in the same experiment to control positive and negative ion fluxes into cells. In addition, the luminescence sensors will be applied to high-throughput screening of autofluorescent neuroactive compounds. In addition to these in vitro applications, however, the primary goal of this project is to apply these novel sensors for the first time to several new in vivo applications of measuring neural activity in a minimally invasive manner in freely behaving rodents. This project is appropriate for the R21 Exploratory/Developmental Research Grant mechanism of the NIMH because it will develop new technologies and tools to advance spatiotemporal analyses of complex circuits and cellular interactions in the brains of multiple model animal species.